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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:117002332+117002427 96bp ACGTGTGCACCTTGTATGTG GGTAGGGGAGGGTGCTAAAG
Mutant= 96 bp
Wild Type = 96 bp
Wt Sequence: acgtgtgcaccttgtatgtgcacgtgcacacacatacActtgggtgcataacacccggaggcaagaatctttacttctttagcaccctcccctacc
Mut Sequence: acgtgtgcaccttgtatgtgcacgtgcacacacatacACttggaagtatttgctgtctgcacagcacgctcttccagaggctgatgggaaaagtgt
A 469 bp deletion beginning at Chromosome 10 position 117,002,370 bp and ending after 117,002,838 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66270 | ACG TGT GCA CCT TGT ATG TG | Common | A | |||
66271 | GGT AGG GGA GGG TGC TAA AG | Wild type Reverse | A | |||
66272 | ACA CTT TTC CCA TCA GCC TCT | Mutant Reverse | A | |||
66273 | Fluorophore-1 | TGC ACA CAC ATA CAC TTG GAA GTA T | Quencher-1 | MUT Probe | ||
66274 | Fluorophore-2 | CAT AAC ACC CGG AGG CAA | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66270 | 0.40 uM |
66271 | 0.40 uM |
66272 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |